Project: Summary/Abstract: Immune Monitoring and Flow Cytometry The Immune Monitoring and Flow Cytometry (IMFC) Shared Resource, also known as DartLab, arose from the fusion of immune monitoring and flow cytometry services. Immune monitoring provides for specimen processing, immune monitoring, customized immunoassays, maintenance of a repository of cryopreserved heathy donor leukocytes, consultation on experimental design, and data analysis. Flow cytometry services include provision of flow cytometers for unassisted and assisted use, cell sorting, a flow cytometry course, and instrument training. Although not CLIA-certified, we follow CLIA-recommended guidelines for high-complexity testing for QA/QC of our instruments, equipment, and immunoassays. New equipment since the previous cycle includes an XFe96 extracellular flux analyzer (Seahorse), purchased in 2017 with funds provided by NCCC. Two new 5-laser analyzers have enabled us to design 16-color panels for human B cells and monocytes/DCs to investigate the CD40/CD40L signature of these cells. IMFC?s impact is exemplified by its broad use. The Shared Resource has been used in the current funding period by a total of 71 PIs, including 60 NCCC members (85% of total users). Forty-one (58%) were NCCC members with peer-review funded projects, and we are requesting only 5% of the Total IMFC Budget from CCSG support. Users now are aligned with all four NCCC Programs (55 current users). Non-NCCC Member users are from eight Dartmouth College departments, and 9 Pharma users, including 3 Dartmouth biotech start-ups. Of the 60 NCCC members that used IMFC services, 95% used the flow cytometer analyzers (mainly unassisted) and cell sorter; 39% used multiplexed cytokine assays, either human 41-plex or mouse 32-plex assays that are purchased per well; and 24% (12 PIs including 7 NCCC clinicians) used specimen processing and/or immune monitoring services. With regard to the NCCC Immunology & Cancer Immunotherapy (ICI) Program studies of tissue-resident memory T cells, IMFC trained the lab students in flow cytometry principles and practices, including multi-color panel design and how to use a flow cytometer analyzer; and IMFC furnished the MACSQuant-10 analyzer used for these studies and consulted on panel design and optimization. For evaluating immune checkpoint inhibitors, IMFC performed longitudinal patient specimen processing and immune monitoring, designed and optimized panels, stained patient fresh whole blood, performed flow cytometry, and analyzed and interpreted flow cytometry results. IMFC provides an essential flow cytometry service to three of the four NCCC research programs and an essential clinical trial immune monitoring service to clinicians lacking laboratory space. Both services are especially critical to the mission and strategic priorities of NCCC in defining how the immune system impacts cancer development and progression, developing novel strategies to treat cancer, and conducting clinical trials to test novel therapeutics and approaches to improve patient survival.